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Sec16 influences transitional ER sites by regulating rather than organizing COPII.

Bharucha N, Liu Y, Papanikou E, McMahon C, Esaki M, Jeffrey PD, Hughson FM, Glick BS - Mol. Biol. Cell (2013)

Bottom Line: An upstream conserved region (UCR) localizes Sec16 to tER sites.We propose that Sec16 does not in fact organize COPII.Instead, regulation of COPII turnover can account for the influence of Sec16 on tER sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637 Department of Molecular Biology, Princeton University, Princeton, NJ 08544.

ABSTRACT
During the budding of coat protein complex II (COPII) vesicles from transitional endoplasmic reticulum (tER) sites, Sec16 has been proposed to play two distinct roles: negatively regulating COPII turnover and organizing COPII assembly at tER sites. We tested these ideas using the yeast Pichia pastoris. Redistribution of Sec16 to the cytosol accelerates tER dynamics, supporting a negative regulatory role for Sec16. To evaluate a possible COPII organization role, we dissected the functional regions of Sec16. The central conserved domain, which had been implicated in coordinating COPII assembly, is actually dispensable for normal tER structure. An upstream conserved region (UCR) localizes Sec16 to tER sites. The UCR binds COPII components, and removal of COPII from tER sites also removes Sec16, indicating that COPII recruits Sec16 rather than the other way around. We propose that Sec16 does not in fact organize COPII. Instead, regulation of COPII turnover can account for the influence of Sec16 on tER sites.

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Altered dynamics of tER sites in sec16‑P1092L mutant cells. (A) Shrinkage of tER sites after fusion events in sec16‑P1092L cells. A sec16‑P1092L strain expressing Sec13‑GFP was grown at room temperature, then warmed to 36.5°C for ∼45 min before imaging by 4D microscopy at 36.5°C. Shown are merged fluorescence and differential interference contrast (DIC) images of representative cells. The arrowhead marks a pair of tER sites that underwent fusion followed by shrinkage. Frames are taken from Supplemental Movie S2, and the time from the beginning of the movie is shown in minutes:seconds format. Scale bar, 5 μm. (B) Quantitation of tER site shrinkage in wild-type and sec16‑P1092L cells. From 4D movies of the type shown in A, ∼20 newly fused tER sites were chosen at random for WT or sec16‑P1092L cells. The half-times for shrinkage were determined from plots of the type shown in Supplemental Figure S4A. Each dot represents an individual fused tER site, and the horizontal lines represent the average half-times. (C) Quantitation of de novo tER site formation in wild-type and sec16‑P1092L cells. The cells expressed Sec13-GFP, and, where indicated, they also expressed Sar1(T34N) from the inducible AOX1 promoter. Cultures were shifted to inducing methanol medium for 3 h at room temperature, grown at 36.5°C for an additional 50 min, and then imaged by 4D microscopy at 36.5°C for either 40 min for wild-type cells or 10 min for sec16‑P1092L cells. For each culture, the number of de novo tER site formation events was recorded for ∼20 cells. Plotted are the hourly mean and SEM values. (D) Prevention of tER dispersal by expression of Sar1(T34N). Wild-type or sec16‑P1092L cells expressing Sec13-GFP were transformed with a control vector or a vector encoding Sar1(T34N) expressed from an inducible promoter. Cultures were shifted to inducing methanol medium for 2.5 h at room temperature and then grown at 36.5°C for an additional 1 h before imaging. Shown are merged fluorescence and DIC images of representative cells. Scale bar, 5 μm. (E) Quantitation of the results from D. For each of the four conditions, the tER sites were counted in ∼50 cells. Plotted are mean and SEM. (F) Cytosolic localization of Sec16-P1092L in the presence of Sar1(T34N). In a sec16-P1092L strain, the mutant sec16 gene was tagged with GFP by gene replacement. The resulting strain was transformed with a control vector or a vector encoding Sar1(T34N) expressed from an inducible promoter. Induction and imaging were performed as in D. The expression of Sar1(T34N) was confirmed by measuring growth inhibition (Connerly et al., 2005; unpublished data). Scale bar, 5 μm.
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Figure 6: Altered dynamics of tER sites in sec16‑P1092L mutant cells. (A) Shrinkage of tER sites after fusion events in sec16‑P1092L cells. A sec16‑P1092L strain expressing Sec13‑GFP was grown at room temperature, then warmed to 36.5°C for ∼45 min before imaging by 4D microscopy at 36.5°C. Shown are merged fluorescence and differential interference contrast (DIC) images of representative cells. The arrowhead marks a pair of tER sites that underwent fusion followed by shrinkage. Frames are taken from Supplemental Movie S2, and the time from the beginning of the movie is shown in minutes:seconds format. Scale bar, 5 μm. (B) Quantitation of tER site shrinkage in wild-type and sec16‑P1092L cells. From 4D movies of the type shown in A, ∼20 newly fused tER sites were chosen at random for WT or sec16‑P1092L cells. The half-times for shrinkage were determined from plots of the type shown in Supplemental Figure S4A. Each dot represents an individual fused tER site, and the horizontal lines represent the average half-times. (C) Quantitation of de novo tER site formation in wild-type and sec16‑P1092L cells. The cells expressed Sec13-GFP, and, where indicated, they also expressed Sar1(T34N) from the inducible AOX1 promoter. Cultures were shifted to inducing methanol medium for 3 h at room temperature, grown at 36.5°C for an additional 50 min, and then imaged by 4D microscopy at 36.5°C for either 40 min for wild-type cells or 10 min for sec16‑P1092L cells. For each culture, the number of de novo tER site formation events was recorded for ∼20 cells. Plotted are the hourly mean and SEM values. (D) Prevention of tER dispersal by expression of Sar1(T34N). Wild-type or sec16‑P1092L cells expressing Sec13-GFP were transformed with a control vector or a vector encoding Sar1(T34N) expressed from an inducible promoter. Cultures were shifted to inducing methanol medium for 2.5 h at room temperature and then grown at 36.5°C for an additional 1 h before imaging. Shown are merged fluorescence and DIC images of representative cells. Scale bar, 5 μm. (E) Quantitation of the results from D. For each of the four conditions, the tER sites were counted in ∼50 cells. Plotted are mean and SEM. (F) Cytosolic localization of Sec16-P1092L in the presence of Sar1(T34N). In a sec16-P1092L strain, the mutant sec16 gene was tagged with GFP by gene replacement. The resulting strain was transformed with a control vector or a vector encoding Sar1(T34N) expressed from an inducible promoter. Induction and imaging were performed as in D. The expression of Sar1(T34N) was confirmed by measuring growth inhibition (Connerly et al., 2005; unpublished data). Scale bar, 5 μm.

Mentions: Our analysis focused on tER sites that had just undergone fusion and were therefore poised to shrink back to the steady-state size. In wild-type cells the shrinkage of newly fused tER sites was gradual, often requiring 30 min or more to reach completion (Bevis et al., 2002; Supplemental Figure S4A and Supplemental Movie S1). By contrast, in sec16‑P1092L cells the shrinkage was typically complete within 1–2 min (Figure 6A, Supplemental Figure S4A, and Supplemental Movie S2). tER sites in sec16‑P1092L cells sometimes disappeared (Supplemental Figure S4A), a phenomenon that we never observed in wild-type cells. We analyzed ∼20 fusion events for each strain. In wild-type cells the average half-time for shrinkage was 11 min, whereas in sec16‑P1092L cells it was only 20 s (Figure 6B). Faster shrinkage reduced the size of tER sites, which had fluorescence intensities 5.6-fold lower on average in sec16‑P1092L cells than in wild-type cells (Supplemental Figure S4B).


Sec16 influences transitional ER sites by regulating rather than organizing COPII.

Bharucha N, Liu Y, Papanikou E, McMahon C, Esaki M, Jeffrey PD, Hughson FM, Glick BS - Mol. Biol. Cell (2013)

Altered dynamics of tER sites in sec16‑P1092L mutant cells. (A) Shrinkage of tER sites after fusion events in sec16‑P1092L cells. A sec16‑P1092L strain expressing Sec13‑GFP was grown at room temperature, then warmed to 36.5°C for ∼45 min before imaging by 4D microscopy at 36.5°C. Shown are merged fluorescence and differential interference contrast (DIC) images of representative cells. The arrowhead marks a pair of tER sites that underwent fusion followed by shrinkage. Frames are taken from Supplemental Movie S2, and the time from the beginning of the movie is shown in minutes:seconds format. Scale bar, 5 μm. (B) Quantitation of tER site shrinkage in wild-type and sec16‑P1092L cells. From 4D movies of the type shown in A, ∼20 newly fused tER sites were chosen at random for WT or sec16‑P1092L cells. The half-times for shrinkage were determined from plots of the type shown in Supplemental Figure S4A. Each dot represents an individual fused tER site, and the horizontal lines represent the average half-times. (C) Quantitation of de novo tER site formation in wild-type and sec16‑P1092L cells. The cells expressed Sec13-GFP, and, where indicated, they also expressed Sar1(T34N) from the inducible AOX1 promoter. Cultures were shifted to inducing methanol medium for 3 h at room temperature, grown at 36.5°C for an additional 50 min, and then imaged by 4D microscopy at 36.5°C for either 40 min for wild-type cells or 10 min for sec16‑P1092L cells. For each culture, the number of de novo tER site formation events was recorded for ∼20 cells. Plotted are the hourly mean and SEM values. (D) Prevention of tER dispersal by expression of Sar1(T34N). Wild-type or sec16‑P1092L cells expressing Sec13-GFP were transformed with a control vector or a vector encoding Sar1(T34N) expressed from an inducible promoter. Cultures were shifted to inducing methanol medium for 2.5 h at room temperature and then grown at 36.5°C for an additional 1 h before imaging. Shown are merged fluorescence and DIC images of representative cells. Scale bar, 5 μm. (E) Quantitation of the results from D. For each of the four conditions, the tER sites were counted in ∼50 cells. Plotted are mean and SEM. (F) Cytosolic localization of Sec16-P1092L in the presence of Sar1(T34N). In a sec16-P1092L strain, the mutant sec16 gene was tagged with GFP by gene replacement. The resulting strain was transformed with a control vector or a vector encoding Sar1(T34N) expressed from an inducible promoter. Induction and imaging were performed as in D. The expression of Sar1(T34N) was confirmed by measuring growth inhibition (Connerly et al., 2005; unpublished data). Scale bar, 5 μm.
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Figure 6: Altered dynamics of tER sites in sec16‑P1092L mutant cells. (A) Shrinkage of tER sites after fusion events in sec16‑P1092L cells. A sec16‑P1092L strain expressing Sec13‑GFP was grown at room temperature, then warmed to 36.5°C for ∼45 min before imaging by 4D microscopy at 36.5°C. Shown are merged fluorescence and differential interference contrast (DIC) images of representative cells. The arrowhead marks a pair of tER sites that underwent fusion followed by shrinkage. Frames are taken from Supplemental Movie S2, and the time from the beginning of the movie is shown in minutes:seconds format. Scale bar, 5 μm. (B) Quantitation of tER site shrinkage in wild-type and sec16‑P1092L cells. From 4D movies of the type shown in A, ∼20 newly fused tER sites were chosen at random for WT or sec16‑P1092L cells. The half-times for shrinkage were determined from plots of the type shown in Supplemental Figure S4A. Each dot represents an individual fused tER site, and the horizontal lines represent the average half-times. (C) Quantitation of de novo tER site formation in wild-type and sec16‑P1092L cells. The cells expressed Sec13-GFP, and, where indicated, they also expressed Sar1(T34N) from the inducible AOX1 promoter. Cultures were shifted to inducing methanol medium for 3 h at room temperature, grown at 36.5°C for an additional 50 min, and then imaged by 4D microscopy at 36.5°C for either 40 min for wild-type cells or 10 min for sec16‑P1092L cells. For each culture, the number of de novo tER site formation events was recorded for ∼20 cells. Plotted are the hourly mean and SEM values. (D) Prevention of tER dispersal by expression of Sar1(T34N). Wild-type or sec16‑P1092L cells expressing Sec13-GFP were transformed with a control vector or a vector encoding Sar1(T34N) expressed from an inducible promoter. Cultures were shifted to inducing methanol medium for 2.5 h at room temperature and then grown at 36.5°C for an additional 1 h before imaging. Shown are merged fluorescence and DIC images of representative cells. Scale bar, 5 μm. (E) Quantitation of the results from D. For each of the four conditions, the tER sites were counted in ∼50 cells. Plotted are mean and SEM. (F) Cytosolic localization of Sec16-P1092L in the presence of Sar1(T34N). In a sec16-P1092L strain, the mutant sec16 gene was tagged with GFP by gene replacement. The resulting strain was transformed with a control vector or a vector encoding Sar1(T34N) expressed from an inducible promoter. Induction and imaging were performed as in D. The expression of Sar1(T34N) was confirmed by measuring growth inhibition (Connerly et al., 2005; unpublished data). Scale bar, 5 μm.
Mentions: Our analysis focused on tER sites that had just undergone fusion and were therefore poised to shrink back to the steady-state size. In wild-type cells the shrinkage of newly fused tER sites was gradual, often requiring 30 min or more to reach completion (Bevis et al., 2002; Supplemental Figure S4A and Supplemental Movie S1). By contrast, in sec16‑P1092L cells the shrinkage was typically complete within 1–2 min (Figure 6A, Supplemental Figure S4A, and Supplemental Movie S2). tER sites in sec16‑P1092L cells sometimes disappeared (Supplemental Figure S4A), a phenomenon that we never observed in wild-type cells. We analyzed ∼20 fusion events for each strain. In wild-type cells the average half-time for shrinkage was 11 min, whereas in sec16‑P1092L cells it was only 20 s (Figure 6B). Faster shrinkage reduced the size of tER sites, which had fluorescence intensities 5.6-fold lower on average in sec16‑P1092L cells than in wild-type cells (Supplemental Figure S4B).

Bottom Line: An upstream conserved region (UCR) localizes Sec16 to tER sites.We propose that Sec16 does not in fact organize COPII.Instead, regulation of COPII turnover can account for the influence of Sec16 on tER sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637 Department of Molecular Biology, Princeton University, Princeton, NJ 08544.

ABSTRACT
During the budding of coat protein complex II (COPII) vesicles from transitional endoplasmic reticulum (tER) sites, Sec16 has been proposed to play two distinct roles: negatively regulating COPII turnover and organizing COPII assembly at tER sites. We tested these ideas using the yeast Pichia pastoris. Redistribution of Sec16 to the cytosol accelerates tER dynamics, supporting a negative regulatory role for Sec16. To evaluate a possible COPII organization role, we dissected the functional regions of Sec16. The central conserved domain, which had been implicated in coordinating COPII assembly, is actually dispensable for normal tER structure. An upstream conserved region (UCR) localizes Sec16 to tER sites. The UCR binds COPII components, and removal of COPII from tER sites also removes Sec16, indicating that COPII recruits Sec16 rather than the other way around. We propose that Sec16 does not in fact organize COPII. Instead, regulation of COPII turnover can account for the influence of Sec16 on tER sites.

Show MeSH
Related in: MedlinePlus